This study was aimed at understanding the effect of smoking on pRb phosphorylation and the clinicopathological significance of phospho-pRb in non-small cell lung cancers (NSCLCs). Phospho-pRb (Ser-807/811) expression was not detected in 149 (39%) of 382 patients, and the mean phospho-pRb (Ser-807/811) level was 5.7%. Squamous cell carcinoma had higher phospho-pRb (Ser-807/811) levels than adenocarcinoma (7.1%+/-10.4% versus 4.7%+/-7.9%; P=0.003). The association between phospho-pRb (Ser-807/811) levels and exposure to tobacco smoke was different according to the statuses of cyclin D1 expression and p16 methylation, suggesting that their statuses might play a role as an effect modifier in the relationship between phospho-pRb (Ser-807/811) levels and exposure to tobacco smoke. In stratified multivariate analysis, phospho-pRb (Ser-807/811) levels were not associated with exposure to tobacco smoke in 38 patients with p16 hypermethylation and cyclin D1 expression >5%, after adjusting for confounding factors. However, in the remaining 344 patients, the mean phospho-pRb (Ser-807/811) levels in patients who had smoked >40 pack years increased by 4.65% (P<0.0001) on average than those who had never smoked. No association was found between the phospho-pRb (Ser-807/811) levels and overall survival. In conclusion, the present study suggests that exposure to tobacco smoke is associated with phosphorylation of pRb in NSCLC patients and its relationship depends on the p16 methylation status and cyclin D1 expression levels.